Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Perspective
  • Published:

Turning single-molecule localization microscopy into a quantitative bioanalytical tool

Abstract

Single-molecule localization microscopy (SMLM) generates super-resolution images by serially detecting individual fluorescent molecules. The power of SMLM, however, goes beyond images: biologically relevant information can be extracted from the mathematical relationships between the positions of the fluorophores in space and time. Here we review the history of SMLM and how recent progress in methods for spatial point analysis has enabled quantitative measurement of SMLM data, providing insights into biomolecule patterning, clustering and oligomerization in biological systems.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Timeline of SMLM developments, seen as a recapitulation of the SMLM workflow.
Figure 2: Spatial analysis approaches applied to identical SMLM data.
Figure 3: Scheme for kinetics-based molecular counting in SMLM data.

Similar content being viewed by others

References

  1. Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006).

    CAS  PubMed  Google Scholar 

  2. Rust, M.J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793–796 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Hess, S.T., Girirajan, T.P.K. & Mason, M.D. Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys. J. 91, 4258–4272 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Dickson, R.M., Cubitt, A.B., Tsien, R.Y. & Moerner, W.E. On/off blinking and switching behaviour of single molecules of green fluorescent protein. Nature 388, 355–358 (1997).

    CAS  PubMed  Google Scholar 

  5. Dickson, R.M., Norris, D.J., Tzeng, Y.L. & Moerner, W.E. Three-dimensional imaging of single molecules solvated in pores of poly(acrylamide) gels. Science 274, 966–968 (1996).

    CAS  PubMed  Google Scholar 

  6. Funatsu, T., Harada, Y., Tokunaga, M., Saito, K. & Yanagida, T. Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules in aqueous solution. Nature 374, 555–559 (1995).

    CAS  PubMed  Google Scholar 

  7. Thompson, R.E., Larson, D.R. & Webb, W.W. Precise nanometer localization analysis for individual fluorescent probes. Biophys. J. 82, 2775–2783 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Yildiz, A. et al. Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization. Science 300, 2061–2065 (2003).

    CAS  PubMed  Google Scholar 

  9. Dahan, M. et al. Diffusion dynamics of glycine receptors revealed by single-quantum dot tracking. Science 302, 442–445 (2003).

    CAS  PubMed  Google Scholar 

  10. Yu, J., Xiao, J., Ren, X., Lao, K. & Xie, X.S. Probing gene expression in live cells, one protein molecule at a time. Science 311, 1600–1603 (2006).

    CAS  PubMed  Google Scholar 

  11. Douglass, A.D. & Vale, R.D. Single-molecule microscopy reveals plasma membrane microdomains created by protein-protein networks that exclude or trap signaling molecules in T cells. Cell 121, 937–950 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Lakadamyali, M., Rust, M.J., Babcock, H.P. & Zhuang, X. Visualizing infection of individual influenza viruses. Proc. Natl. Acad. Sci. USA 100, 9280–9285 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Lippincott-Schwartz, J. & Patterson, G.H. Photoactivatable fluorescent proteins for diffraction-limited and super-resolution imaging. Trends Cell Biol. 19, 555–565 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Heilemann, M. et al. Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. Angew. Chem. Int. Ed. Engl. 47, 6172–6176 (2008).

    CAS  PubMed  Google Scholar 

  15. Bretschneider, S., Eggeling, C. & Hell, S.W. Breaking the diffraction barrier in fluorescence microscopy by optical shelving. Phys. Rev. Lett. 98, 218103 (2007).

    PubMed  Google Scholar 

  16. Sharonov, A. & Hochstrasser, R.M. Wide-field subdiffraction imaging by accumulated binding of diffusing probes. Proc. Natl. Acad. Sci. USA 103, 18911–18916 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Giannone, G. et al. Dynamic superresolution imaging of endogenous proteins on living cells at ultra-high density. Biophys. J. 99, 1303–1310 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Lew, M.D. et al. Three-dimensional superresolution colocalization of intracellular protein superstructures and the cell surface in live Caulobacter crescentus. Proc. Natl. Acad. Sci. USA 108, E1102–E1110 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Jungmann, R. et al. Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT. Nat. Methods 11, 313–318 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Baddeley, D., Cannell, M.B. & Soeller, C. Visualization of localization microscopy data. Microsc. Microanal. 16, 64–72 (2010).

    CAS  PubMed  Google Scholar 

  21. Williamson, D.J. et al. Pre-existing clusters of the adaptor Lat do not participate in early T cell signaling events. Nat. Immunol. 12, 655–662 (2011).

    CAS  PubMed  Google Scholar 

  22. Lillemeier, B.F. et al. TCR and Lat are expressed on separate protein islands on T cell membranes and concatenate during activation. Nat. Immunol. 11, 543 (2010).

    CAS  Google Scholar 

  23. Rossy, J., Owen, D.M., Williamson, D.J., Yang, Z. & Gaus, K. Conformational states of the kinase Lck regulate clustering in early T cell signaling. Nat. Immunol. 14, 82–89 (2013).

    CAS  PubMed  Google Scholar 

  24. Szymborska, A. et al. Nuclear pore scaffold structure analyzed by super-resolution microscopy and particle averaging. Science 341, 655–658 (2013).

    CAS  PubMed  Google Scholar 

  25. Ehmann, N. et al. Quantitative super-resolution imaging of Bruchpilot distinguishes active zone states. Nat. Commun. 5, 4650 (2014).

    CAS  PubMed  Google Scholar 

  26. Xu, K., Zhong, G. & Zhuang, X. Actin, spectrin, and associated proteins form a periodic cytoskeletal structure in axons. Science 339, 452–456 (2013).

    CAS  PubMed  Google Scholar 

  27. Ricci, M.A., Manzo, C., García-Parajo, M.F., Lakadamyali, M. & Cosma, M.P. Chromatin fibers are formed by heterogeneous groups of nucleosomes in vivo. Cell 160, 1145–1158 (2015).

    CAS  PubMed  Google Scholar 

  28. Biteen, J.S., Goley, E.D., Shapiro, L. & Moerner, W.E. Three-dimensional super-resolution imaging of the midplane protein FtsZ in live Caulobacter crescentus cells using astigmatism. ChemPhysChem 13, 1007–1012 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Bakshi, S., Siryaporn, A., Goulian, M. & Weisshaar, J.C. Superresolution imaging of ribosomes and RNA polymerase in live Escherichia coli cells. Mol. Microbiol. 85, 21–38 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Endesfelder, U. et al. Multiscale spatial organization of RNA polymerase in Escherichia coli. Biophys. J. 105, 172–181 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Gunzenhäuser, J., Olivier, N., Pengo, T. & Manley, S. Quantitative super-resolution imaging reveals protein stoichiometry and nanoscale morphology of assembling HIV-Gag virions. Nano Lett. 12, 4705–4710 (2012).

    PubMed  Google Scholar 

  32. Pham, S. et al. Cryo-electron microscopy and single molecule fluorescent microscopy detect CD4 receptor induced HIV size expansion prior to cell entry. Virology 486, 121–133 (2015).

    CAS  PubMed  Google Scholar 

  33. Pereira, C.F., Rossy, J., Owen, D.M., Mak, J. & Gaus, K. HIV taken by STORM: super-resolution fluorescence microscopy of a viral infection. Virol. J. 9, 84 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Malkusch, S., Muranyi, W., Müller, B., Kräusslich, H.G. & Heilemann, M. Single-molecule coordinate-based analysis of the morphology of HIV-1 assembly sites with near-molecular spatial resolution. Histochem. Cell Biol. 139, 173–179 (2013).

    CAS  PubMed  Google Scholar 

  35. Dempsey, G.T., Vaughan, J.C., Chen, K.H., Bates, M. & Zhuang, X.W. Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging. Nat Methods 8, 1027–1036 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Fernández-Suárez, M. & Ting, A.Y. Fluorescent probes for super-resolution imaging in living cells. Nat. Rev. Mol. Cell Biol. 9, 929–943 (2008).

    PubMed  Google Scholar 

  37. Chozinski, T.J., Gagnon, L.A. & Vaughan, J.C. Twinkle, twinkle little star: photoswitchable fluorophores for super-resolution imaging. FEBS Lett. 588, 3603–3612 (2014).

    CAS  PubMed  Google Scholar 

  38. Shcherbakova, D.M., Sengupta, P., Lippincott-Schwartz, J. & Verkhusha, V.V. Photocontrollable fluorescent proteins for superresolution imaging. Annu. Rev. Biophys. 43, 303–329 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Olivier, N., Keller, D., Gönczy, P. & Manley, S. Resolution doubling in 3D-STORM imaging through improved buffers. PLoS One 8, e69004 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Olivier, N., Keller, D., Rajan, V.S., Gönczy, P. & Manley, S. Simple buffers for 3D STORM microscopy. Biomed. Opt. Express 4, 885–899 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Kaplan, C. & Ewers, H. Optimized sample preparation for single-molecule localization-based superresolution microscopy in yeast. Nat. Protoc. 10, 1007–1021 (2015).

    CAS  PubMed  Google Scholar 

  42. Huang, Z.L. et al. Localization-based super-resolution microscopy with an sCMOS camera. Opt. Express 19, 19156–19168 (2011).

    PubMed  Google Scholar 

  43. Pertsinidis, A. et al. Ultrahigh-resolution imaging reveals formation of neuronal SNARE/Munc18 complexes in situ. Proc. Natl. Acad. Sci. USA 110, E2812–E2820 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Pertsinidis, A., Zhang, Y. & Chu, S. Subnanometre single-molecule localization, registration and distance measurements. Nature 466, 647–651 (2010).

    CAS  PubMed  Google Scholar 

  45. Small, A. & Stahlheber, S. Fluorophore localization algorithms for super-resolution microscopy. Nat. Methods 11, 267–279 (2014).

    CAS  PubMed  Google Scholar 

  46. Deschout, H. et al. Precisely and accurately localizing single emitters in fluorescence microscopy. Nat. Methods 11, 253–266 (2014).

    CAS  PubMed  Google Scholar 

  47. Sage, D. et al. Quantitative evaluation of software packages for single-molecule localization microscopy. Nat. Methods 12, 717–724 (2015).

    CAS  PubMed  Google Scholar 

  48. Clark, P.J. & Evans, F.C. Distance to nearest neighbor as a measure of spatial relationships in populations. Ecology 35, 445–453 (1954).

    Google Scholar 

  49. Veatch, S.L. et al. Correlation functions quantify super-resolution images and estimate apparent clustering due to over-counting. PLoS One 7, e31457 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Annibale, P., Vanni, S., Scarselli, M., Rothlisberger, U. & Radenovic, A. Identification of clustering artifacts in photoactivated localization microscopy. Nat. Methods 8, 527–528 (2011).

    CAS  PubMed  Google Scholar 

  51. Annibale, P., Vanni, S., Scarselli, M., Rothlisberger, U. & Radenovic, A. Quantitative photo activated localization microscopy: unraveling the effects of photoblinking. PLoS One 6, e22678 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Ripley, B.D. Point processes of stochastic geometry. Adv. Appl. Probab. 9, 434–435 (1977).

    Google Scholar 

  53. Owen, D.M. et al. PALM imaging and cluster analysis of protein heterogeneity at the cell surface. J. Biophotonics 3, 446–454 (2010).

    CAS  PubMed  Google Scholar 

  54. Sengupta, P. et al. Probing protein heterogeneity in the plasma membrane using PALM and pair correlation analysis. Nat. Methods 8, 969–975 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Sengupta, P. & Lippincott-Schwartz, J. Quantitative analysis of photoactivated localization microscopy (PALM) datasets using pair-correlation analysis. BioEssays 34, 396–405 (2012).

    PubMed  PubMed Central  Google Scholar 

  56. Subach, F.V. et al. Photoactivatable mCherry for high-resolution two-color fluorescence microscopy. Nat. Methods 6, 153–159 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. Kapoor-Kaushik, N. et al. Distinct mechanisms regulate Lck spatial organization in activated T cells. Front. Immunol. 7, 83 (2016).

    PubMed  PubMed Central  Google Scholar 

  58. Kiskowski, M.A., Hancock, J.F. & Kenworthy, A.K. On the use of Ripley's K-function and its derivatives to analyze domain size. Biophys. J. 97, 1095–1103 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. Rossy, J., Cohen, E., Gaus, K. & Owen, D.M. Method for co-cluster analysis in multichannel single-molecule localisation data. Histochem. Cell Biol. 141, 605–612 (2014).

    CAS  PubMed  Google Scholar 

  60. Georgieva, M. et al. Nanometer resolved single-molecule colocalization of nuclear factors by two-color super resolution microscopy imaging. Methods 105, 44–55 (2016).

    CAS  PubMed  Google Scholar 

  61. Malkusch, S. et al. Coordinate-based colocalization analysis of single-molecule localization microscopy data. Histochem. Cell Biol. 137, 1–10 (2012).

    CAS  PubMed  Google Scholar 

  62. Deschout, H., Shivanandan, A., Annibale, P., Scarselli, M. & Radenovic, A. Progress in quantitative single-molecule localization microscopy. Histochem. Cell Biol. 142, 5–17 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. Getis, A. & Franklin, J. Second-order neighborhood analysis of mapped point patterns. Ecology 68, 473–477 (1987).

    Google Scholar 

  64. Rubin-Delanchy, P. et al. Bayesian cluster identification in single-molecule localization microscopy data. Nat. Methods 12, 1072–1076 (2015).

    CAS  PubMed  Google Scholar 

  65. Ester, M., Kriegel, H.-P., Sander, J. & Xu, X. A density-based algorithm for discovering clusters in large spatial databases with noise. Second International Conference on Knowledge Discovery and Data Mining (KDD-96) 96, 226–231 (1996).

    Google Scholar 

  66. Ankerst, M., Breunig, M.M., Kriegel, H.P. & Sander, J. OPTICS: ordering points to identify the clustering structure. in Proceedings of the 1999 ACM SIGMOD International Conference on Management of Data 49–60 (ACM, 1999).

  67. Achtert, E., Bohm, C. & Kroger, P. DeLiClu: Boosting robustness, completeness, usability, and efficiency of hierarchical clustering by a closest pair ranking. in Proceedings of the 10th Pacific-Asian Conference on Advances in Knowledge Discovery and Data Mining. (PAKDD'06), Singapore, 2006 119–128 (Springer, 2006).

  68. Andronov, L., Orlov, I., Lutz, Y., Vonesch, J.L. & Klaholz, B.P. ClusterViSu, a method for clustering of protein complexes by Voronoi tessellation in super-resolution microscopy. Sci. Rep. 6, 24084 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  69. Levet, F. et al. SR-Tesseler: a method to segment and quantify localization-based super-resolution microscopy data. Nat. Methods 12, 1065–1071 (2015).

    CAS  PubMed  Google Scholar 

  70. Jungmann, R. et al. Quantitative super-resolution imaging with qPAINT. Nat. Methods 13, 439–442 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  71. Nan, X. et al. Single-molecule superresolution imaging allows quantitative analysis of RAF multimer formation and signaling. Proc. Natl. Acad. Sci. USA 110, 18519–18524 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  72. Lee, S.H., Shin, J.Y., Lee, A. & Bustamante, C. Counting single photoactivatable fluorescent molecules by photoactivated localization microscopy (PALM). Proc. Natl. Acad. Sci. USA 109, 17436–17441 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  73. Rollins, G.C., Shin, J.Y., Bustamante, C. & Pressé, S. Stochastic approach to the molecular counting problem in superresolution microscopy. Proc. Natl. Acad. Sci. USA 112, E110–E118 (2015).

    CAS  PubMed  Google Scholar 

  74. Puchner, E.M., Walter, J.M., Kasper, R., Huang, B. & Lim, W.A. Counting molecules in single organelles with superresolution microscopy allows tracking of the endosome maturation trajectory. Proc. Natl. Acad. Sci. USA 110, 16015–16020 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  75. Shivanandan, A., Unnikrishnan, J. & Radenovic, A. Accounting for limited detection efficiency and localization precision in cluster analysis in single molecule localization microscopy. PLoS One 10, e0118767 (2015).

    PubMed  PubMed Central  Google Scholar 

  76. Avilov, S. et al. In cellulo evaluation of phototransformation quantum yields in fluorescent proteins used as markers for single-molecule localization microscopy. PLoS One 9, e98362 (2014).

    PubMed  PubMed Central  Google Scholar 

  77. Lando, D. et al. Quantitative single-molecule microscopy reveals that CENP-A(Cnp1) deposition occurs during G2 in fission yeast. Open Biol. 2, 120078 (2012).

    PubMed  PubMed Central  Google Scholar 

  78. Legant, W.R. et al. High-density three-dimensional localization microscopy across large volumes. Nat. Methods 13, 359–365 (2016).

    PubMed  PubMed Central  Google Scholar 

  79. Venkataramani, V., Herrmannsdörfer, F., Heilemann, M. & Kuner, T. SuReSim: simulating localization microscopy experiments from ground truth models. Nat. Methods 13, 319–321 (2016).

    PubMed  Google Scholar 

  80. Durisic, N. et al. Stoichiometry of the human glycine receptor revealed by direct subunit counting. J. Neurosci. 32, 12915–12920 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  81. Burgert, A., Letschert, S., Doose, S. & Sauer, M. Artifacts in single-molecule localization microscopy. Histochem. Cell Biol. 144, 123–131 (2015).

    CAS  PubMed  Google Scholar 

  82. Wolter, S., Endesfelder, U., van de Linde, S., Heilemann, M. & Sauer, M. Measuring localization performance of super-resolution algorithms on very active samples. Opt. Express 19, 7020–7033 (2011).

    PubMed  Google Scholar 

  83. Plowman, S.J., Muncke, C., Parton, R.G. & Hancock, J.F. H-ras, K-ras, and inner plasma membrane raft proteins operate in nanoclusters with differential dependence on the actin cytoskeleton. Proc. Natl. Acad. Sci. USA 102, 15500–15505 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  84. Belov, V.N., Wurm, C.A., Boyarskiy, V.P., Jakobs, S. & Hell, S.W. Rhodamines NN: a novel class of caged fluorescent dyes. Angew. Chem. Int. Ed Engl. 49, 3520–3523 (2010).

    CAS  PubMed  Google Scholar 

  85. Mitchison, T.J., Sawin, K.E., Theriot, J.A., Gee, K. & Mallavarapu, A. Caged fluorescent probes. Methods Enzymol. 291, 63–78 (1998).

    CAS  PubMed  Google Scholar 

  86. Lavis, L.D. et al. Bright photoactivatable fluorophores for single-molecule imaging. Nat. Methods 13, 985–988 (2016).

    PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Contributions

P.R.N., D.M.O. and K.G. wrote the manuscript. P.R.N. prepared the figures.

Corresponding author

Correspondence to Katharina Gaus.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Nicovich, P., Owen, D. & Gaus, K. Turning single-molecule localization microscopy into a quantitative bioanalytical tool. Nat Protoc 12, 453–460 (2017). https://doi.org/10.1038/nprot.2016.166

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2016.166

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing